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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2017 - November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 july 2016
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
July 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt
EC Number:
947-738-8
Molecular formula:
Molecular formulas of exemplary components: C33H68KO10P C49H100KO16P C24H49K2O10P C24H50KO10P C16H34KO4P C8H17K2O4P C8H18KO4P
IUPAC Name:
Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal age at the start of the treatment period: approx. 14-15 weeks old. Females were nulliparous and non-pregnant.
Body weight at the allocation of the animals to the experimental groups: males 338-370 g (mean 355.73 g); females 226-253 g (mean 240.20 g)

Housing and feeding:
Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals will be housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females will be housed in groups of 2 in tpe III IV polysulphone cages and during post-mating period for males depending on the mating status. During mating period males and females will be housed together in ratio 1:1 (male to female). After the confirmation of mating, females will be kept individually during gestation/lactation period in type III H, polysulphone cages and males will be returned to their original cage. In each cage Altromin saw fibre will be used as bedding.
- Nesting material provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad injectionem, Delta Medica
Details on exposure:
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.


On days 1 and 2, due to technical error half of female animals were dosed with 5 mL/kg. For compensation the pre-mating period of female animals was extended from 14 to 15 days.

Concentrations in vehicle:
LD 13.34 mg/mL test item (12 mg/mL active ingredient)
MD 33.24 mg/mL test item (30 mg/mL active ingredient)
HD 83.33 mg/mL test item (75 mg/mL active ingredient)
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
According to the study the test item was homogenous (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples) were taken.
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were stored at < -20 °C before they were shipped and analysed. The B-samples were retained at -15 to -35 °C at test facility and discarded after completion of the final study report.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle for 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating for males and 15 days of pre-mating for females and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period for 29 days.
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
per active ingredient (90%, conversion factor 1.1)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
per active ingredient (90%, conversion factor 1.1)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
per active ingredient (90%, conversion factor 1.1)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were selected based on previous dose range finding study. The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Inadvertently, they were not performed in week 5 on male animals.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.


BODY WEIGHT: Yes
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.
Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Litter observations:
The duration of gestation was recorded and calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.
Postmortem examinations (parental animals):
All surviving males were sacrificed on study day 30 (a day after the last dosing) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.

Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.

Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral- buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.

Organ weights
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals euthanised for animal welfare reasons were not recorded.
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Organs weighed at necropsy:
testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), kidneys (paired weight), adrenal (paired weight), pituitary gland, uterus with cervix, ovaries (paired weight), thymus, liver, spleen, brain, heart

The following tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol:
adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, femur with knee joint (no hist-path), Harderian glands (no hist-path), heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (axillary, no hist-path), lymph nodes (mandibular), lymph nodes (mesenteric), mammary gland area (male and female, no hist-path), oesophagus (no hist-path), optic nerves (no hist-path), ovaries, oviducts (no hist-path), pancreas (no hist-path), pituitary (no hist-path), prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular, no hist-path), sciatic nerve, skeletal muscle, skin (no hist-path), spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue (no hist-path), trachea, ureters (no hist-path), urinary bladder, uterus with cervix and vagina, head (no hist-path), nasal cavities (no hist-path).

All animals intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.

A full histopathology was carried out on the preserved organs and tissues mentioned above (except where it has been otherwise stated) of all selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for stomach, liver, adrenal glands, thymus and lung of male and female animals and kidneys of male animals as treatment-related changes were observed in the high dose group.
A full histopathology was carried out on the preserved organs and tissues of all animals which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Immunohistochemistry of α2µ-globulin was performed on kidney of male animals sacrificed at the end of the treatment period of the study (21 animals, 5 randomly selected male).

Immunohistochemistry was performed with Rat α2µ-Globulin Antibody. The specific staining was performed with e.g. Chromotrope-Aniline Blue (details to be reported). The image analysis was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x20. The positive area on the total area was measured in mm2 and calculated as % on the total area. The relative values of positive structures (α2µ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum).
Postmortem examinations (offspring):
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Statistics:
The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.

The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.

The gestation length, pre-coital interval, the number of live births and pre- and post- implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, AGD, litter size and litter weights were summarized.

Parameters like body weight gain and food consumption were calculated as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).
Reproductive indices:
The gestation length, pre-coital interval, the number of live births and pre- and post- implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, AGD, litter size and litter weights were calculated.
Offspring viability indices:
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals sacrificed for animal welfare reasons:
On PMD 15 LD group female was seen with moderate piloerection, moderate sunken flanks, abnormal breathing, pale skin and hypothermia and was euthanised.
MD group female was sacrificed in a moribund condition on GD 20, showing moderate piloerection, moderately reduced spontaneous activity. HD female was seen with severe abnormal breathing, moderate piloerection, severe increased salivation and muscular hypotonia on PMD 13.
HD male was sacrificed on PND 4 in a moribund condition with moderate piloerection, reduced spontaneous activity, half eyelid-closure, abnormal breathing, nasal discharge and moderate salivation.

All observations (including animals euthanised for welfare reasons):
Piloerection was noted on different study days for 1/10 LD group females, 5/10 MD females and 2/10 HD females; as well as 2/10 HD males.
Nasal discharge was seen in 2 HD group males.
Abnormal breathing before application was noted on different study days in 2/10 LD females, 1/10 MD female. 1/10 LD group, 4/10 MD group and 2/10 HD group females and 1/10 LD group males and 1/10 HD group males were seen with abnormal breathing after application on different study days.
On different study days 1/10 LD group females, 4/10 MD group females and 1/10 HD group females regurgitated test item. All animals recovered afterwards except for female which was euthanized due to animal welfare reasons 3 days after regurgitation.
On several study days, female animals of the control, LD, MD and HD group and males of the LD, MD and HD group were observed moving the bedding with the nose or/and salivating immediately after administration. These signs are not assumed to be a sign of systemic toxicity.
Two females of the control group and one female of the MD group had alopecia on different study days and during different time periods. Alopecia was also noted in 1/10 LD group and 1/10 MD group males on different study dates.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One HD male was sacrificed on premating day 4 moribund for animal welfare reasons. LD female was sacrificed moribund on the last day of premating (PMD 15). MD female was sacrificed moribund on GD 20. HD female was sacrificed in a moribund condition on PMD 13.
All animals showed lung affections deemed to be due to accidental aspiration of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Throughout the study the mean body weight of the male animals of the HD group was slightly lower than in the control (5.7 % lower than control at the end of treatment). Overall mean body weight gain was slightly and significantly (p<0.01) lower in the HD group but with high inter individual differences; as 3 out of 9 HD males lost weight and the others (6 animals) gained weight during the study. This is considered test item related.

The mean weight of the HD females during the treatment period was slightly higher than seen in the controls gaining significance (p<0.01) at gestation days 0 and 14. As body weight gain was in the normal range of biological variation this effect is not considered to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A tendency towards lower food consumption was seen in male and female animals of the HD group during the first pre-mating week. This is not considered toxicologically relevant. Besides, there were no considerable differences in food intake between dose groups and control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mean neutrophil rate of male animals in the LD, MD and HD were slightly but dose dependently higher than control. The mean values of the HD male animals were statistically significantly (p<0.01) higher than seen in the control group (24.3 % vs. 12.4 % in the control group). In the HD group this is based on higher neutrophil rate observed in two animals (no. 37 and no. 39), indicating a slight inflammatory process. This correlated with a slight but statistically significantly lower rate of lymphocytes in the HD group (72.4 % vs. 84.3 % in the control group).
This tendency towards a higher neutrophil rate can also be seen in female animals.

A slight but statistically significantly lower mean haemoglobin and haematocrit was observed in male but not female animals of the HD group (14.7 % vs. 16.2 % and 45.4 % vs. 49.6 % in the control group, respectively). Slightly less prominently this was was also observed in males of the MD group (15.1 % vs. 16.2 % and 46.0 % vs. 49.6 % in the control group, respectively). At the same time only a tendency towards lower RBC count was seen. Individual parameters were well in the range of biological variation and without any clinical or histological association this is not considered to be test item related.

Slightly but statistically significantly higher mean platelet count of the LD and HD group male animals is not considered biologically relevant. In the absence of dose- dependency this is also not assumed to be test item related.

Besides, there were no considerable differences in haematology parameters between dose groups and control group.

Treatment with the test item had no effect on blood coagulation parameters at the end of the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Results will be provided after the study is completed.Mean cholesterol values of the HD group males and females and MD group males were slightly but statistically significantly lower than in controls. Due to its slight degree this decrease is not considered to be toxicologically relevant.

Slight but statistically significantly higher mean total protein and albumin values in males of the LD group are not considered toxicologically relevant. Without any dose dependency and histological correlation this is deemed to be incidental.

Liver markers of hepatotoxicity (ALAT and ASAT) were increased in female animal no. 74 of the HD group. This was an isolated finding as liver parameters were normal in the remaining male and female animals of this group.

Besides, there were no considerable differences in clinical biochemistry between dose groups and control group.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Decedents:
In the lungs from all animals, there was an alveolar foreign material indicating test item aspiration. In the lungs from animal from group 2, 3 and 4 there was a chronic multifocal interstitial inflammation. In addition, in the lungs of HD animal, there was granuloma formation. In addition, the forestomach was affected by hyperkeratosis and epithelial hyperplasia and/or inflammation in HD animals. Furthermore, a slight hepatocellular necrosis in the liver from HD male was a contributing factor to poor condition.

Stomach:
An irregular forestomach in three group 4 males was deemed to be test item-related and correlated to inflammatory forestomach lesions. The histological correlate was characterized by epithelial hyperplasia, hyperkeratosis and/or inflammation in animals from all test item-treated groups, whereby in the male at 120 mg/kg, there was only one case of a minimal hyperkeratosis. The latter is considered a lesion within the range of spontaneous background alterations due to its isolated appearance. Furthermore, there were single cases of forestomach erosion in animals at 300 and 750 mg/kg.

Liver:
Hepatocellular hypertrophy, (mainly centrilobular), was noted in all test item-treated groups with a dose-dependent increase in incidence. In one decedent male at 750 mg/kg, there was a slight focal hepatocellular necrosis.

Kidney:
Hyaline inclusions in tubular cells of males increased in severity mainly at 750 mg/kg. At this dose, there was also a case of minimal granular casts, indicating tubular cell necrosis. Tubular basophilia did not increase by dose, and the incidences are considered to be within the range of control alterations. The immunohistochemical evaluation revealed statistically significantly increased values for α2-microglobulin for all test item-treated groups with highest values in group 4.

Adrenal Gland:
In females at 120 and 300 mg/kg, and in both sexes at 750 mg/kg, there was a diffuse cortical hypertrophy.

Thymus:
Thymus atrophy increased in incidence and severity in test item-treated groups.

Peyer’s Patches:
In single animals at 750 mg/kg, there was a minor atrophy of the Peyer’s patches.

Other Findings:
There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change.
Female of group 3 was not pregnant. In this animal, but also in its mating partner male, there were no abnormalities in the reproductive organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Organ:
kidney
liver

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group.

High values for pup mortality in the LD group between PND 0 and PND 4 can be correlated to the litter of dam no. 59 which lost 3 pups between PND 1 and PND 2. However these values are in the normal range of biological variation, do not follow any dose dependency and are not deemed to be test item related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean pup weight on PND 0 was slightly affected in the treatment groups, following a dose dependency. Mean pup weights were significantly lower in the LD (p<0.05, -11 %), MD (p<0.01, -15 %) and HD (p<0.001, -20 %) groups when compared to controls. On PND 4 only the mean pup weight of the HD was significant lower than control (p<0.05, -15 %). On PND 13 mean pup weight did not differ between the groups considerably. The mean total litter weight on PND 0, 4 and 13 was marginally and statistically non-signifciantly lower in the HD group than in the control group (PND 0 -28 %, PND 4 -26 %, PND 13 -22 %). As values were in the range of historical control data this effect is considered to be test item related but not adverse. Additionally, given the adverse findings in dams and the absence of any sign of developmental toxicity apart from weight effects, it can be assumed these finings are related ather to maternal toxicity.

The lower number of female pups and slightly lower number of female pups on PND 0, 4 and 13 of the HD group resulted in a significantly lower female litter weight on corresponding days (PND 0 -46 %, p<0.05; PND 4, -44 %, p<0.05; PND 13 -47 %, p<0.05). This is considered to be a test item related effect at the HD level.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Test item had no effect on serum T4 level of PND 13 pup.
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
A slight, statistical significant lower mean pup weight on PND 0 for male and female pups were observed in the LD, MD and HD group resulting in a slight and significant lower mean cube root of pup weight.
The anogenital distance of pups of male animals was comparable in all groups. AGD is always correlated with cube root of pup body weight, litter size and sex ratio (for data normalization to simulate linear measurement). The relative anogenital distance of male pups was slightly but significantly (p<0.05) higher in the LD and HD group when compared with the control. However, values were in the normal range of variations and therefore not considered to be test item related. The mean anogenital distance of the female pups of the MD and HD group was significant lower than in the control (MD p<0.001, HD p<0.05). However, the relative anogenital distance of the female pups of the MD group was only slightly though statistically significantly (p<0.01) lower for the MD group only. This is not considered to be test item related.
The nipple retention on PND 12 was significant higher in the LD and MD group, when compared to the control (p<0.05). As all values were in the normal range of biological variation and values did not follow any dose dependency, this effect was not considered to be test item related.

Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item had no effect on pup thyroid gland weight of PND 13 pup. However, mean weight of the thyroid/parathyroid gland of the female pups was statistically significantly increased in the MD group (145 % of the control). As this was not associated with a significantly increased serum T4 and histopathological findings and values were within the normal range of historical control data, this is not assumed to be toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like blue snout or blue head on PND 0 (dam of the LD group); dark skin area on neck PND 0 or dark snout on PND 0 (dam of the MD group); no indication of suckling on PND 0 (dam of the HD group); dark anus on PND 0 (dam of the HD group) and blue feet on single pups. Dam of the HD group had 2 thin pups on PND 0 which survived until the end of the scheduled treatment period and 3 pups with flattened abdomen on PND 0 which were found dead on PND 0.
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 750 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change. No findings were noted in the reproductive organs of non-pregnant females and the mating partner males.

A slightly higher mean post-implantation loss observed at the high dose level is considered an isolated finding. In the absence of other correlating contributing factors for effect on developmental and reproductive toxicity in terms of effect on parental histopathology, pup weight, pup survival, reproductive and developmental indices, hormone analysis, estrus cyclicity, the NOAEL for this reproduction/developmental toxicity screening study may be considered to be 750 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
The NOAEL for this reproduction/developmental toxicity screening study may be considered to be 750 mg/kg bw/day.
Executive summary:

The NOAEL for this reproduction/developmental toxicity screening study may be considered to be 750 mg/kg bw/day.

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